1. Field of the Invention
The present invention relates to methods for producing a fraction of an even number saccharide or odd number saccharide having a chondroitin structure.
2. Description of the Related Art
The abbreviations used in the present specification are mentioned first.
CH: Chondroitin
CS: Chondroitin sulfate
GlcA: Glucuronic acid
GalNAc: N-Acetylgalactosamine
HA: Hyaluronic acid
K4CP: Chondroitin polymerase derived from the Escherichia coli K4 strain
MALDI-TOF-MS: Matrix assisted laser desorption/ionization time of flight mass spectrometry
UDP: Uridine-5′-diphosphate
CH is one type of glycosaminoglycans, which comprises GlcA and GalNAc residues alternately and linearly linked with β1-3 linkages and β1-4 linkages. CH exists in cartilages and many of connective tissues in animal living bodies as chondroitin sulfate proteoglycans, and plays important roles in cell adhesion, development, differentiation, nerve cell extension, chondrogenesis and osteogenesis, anagenesis, and so forth. CS is marketed as a substance useful for drugs, health food, and so forth.
The reducing end of CH is usually bound to a core protein of a proteoglycan, and repetition disaccharide units of GlcA-GalNAc are linked to a serine residue in the protein via the so-called linkage region tetrasaccharide consisting of xylose-galactose-galactose-GlcA. However, the non-reducing end thereof has not been identified yet. Although commercially available CS is considered to have a GalNAc residue as the reducing end and a GlcA residue as the non-reducing end in many cases with acid treatment, enzyme treatment and so forth used for the production thereof, the non-reducing end may differ depending on production lots, and the present situation is that products containing CS molecules having different structures of non-reducing ends are marketed.
Although an animal-derived CH polymerase has been cloned, this enzyme itself has no CH synthesis activity, and even if the activity is exhibited, the activity is weak. Therefore, it is not sufficient for efficient industrial production of CH saccharide chain. Further, K4CP has also been cloned, and it is known that this enzyme shows a CH synthesis activity by itself, and CH can be efficiently produced by using this enzyme (Patent document 1, Non-patent document 1). However, control of the non-reducing end saccharide residue of CH, control of content ratio of even number saccharides and odd number saccharides in a product, and so forth with use of K4CP have not been described nor suggested.    Patent document 1: Japanese Patent Laid-open Publication (KOKAI) No. 2003-199583    Non-patent document 1: Ninomiya, T. et al., 2002, Journal of Biological Chemistry, Vol. 277, No. 24, pp. 21567-21575